The present invention relates to nucleic acid detection methods, in particular to quantitative nucleic acid detection methods.
Detection, especially quantitative detection of a particular nucleic acid sequence, as an indication of the presence of an organism e.g. a pathogen in a clinical sample or a contaminant in a food or environmental sample, such as toxin-producing cyanobacteria in water sources, or of mRNA to show a change in transcription levels, is a valuable microbiological tool. In addition, in the diagnostic or forensic use of nucleic acid analysis or in the study of polymorphisms, full sequencing of the target nucleic acid may be unnecessary where the detection of a single base variation or mismatch is sufficient to give a positive identification. Such a single base variation or mismatch may, for example, arise from allelic variation or polymorphism, a point mutation, or any deletion or insertion of genetic material where the detection of a single abnormal or species specific base will give the required information.
Through our work in the development of methods for the detection of bacteria in water, we have developed a new nucleic acid detection method suitable for a wide variety of applications in the environmental, agricultural, food, veterinary, health and medical fields and indeed as a general tool in molecular biology.
There are a number of techniques available for the analysis of nucleic acids including the manufacture of synthetic oligonucleotide probes, particularly labelled probes, for hybridisation to target sequences; in vitro amplification of target nucleic acid sequences by PCR and other related amplification methods and (automated) direct DNA sequencing. These have led to the development of novel approaches for the detection and characterisation of nucleic acids in environmental monitoring (Bej, A. K. and Mahbubani, M. H. [1994] in PCR Technology: Current Innovations p 327-339 and Bowman, J. P. and Sayler, G. S. [1996] in Molecular approaches to environmental microbiology p 63-97). There are three main strategies for quantification of amplified DNA; i.e. size separation by electrophoresis, hybridization to capture probes, and real-time detection. The problems with the gel electrophoresis method are the detection of multiple targets in a single reaction, and interpretation of the results. Size separation detection of multiplex amplifications is also difficult to achieve because the amplification ratios of amplicons with different sizes are dependent on DNA quality.
The capture probe assay is based on hybridization of the entire amplified fragments. Evidently, this assay is not suitable for separation and quantification of homologous amplicons, e.g. products of competitive amplifications. The different amplicons will form sandwich hybridizations at the homologous sites, leading to the capture of both target and non-target fragments, even if the capture site is discriminating.
The ABI PRISM(trademark) 7700 Sequence Detection System (Perkin Elmer, Foster City, Calif.) provides real-time quantitative PCR amplification. However, multiplex assays are limited by the number of fluorochromes available and their overlapping fluorescent spectra.
It is always an aim in nucleic acid detection methods to increase their specificity, ie. to reduce non-specific binding or detection of a non-specific background signal. A separate but associated goal is to increase the sensitivity of the detection method, ie. to allow measurement of very small amounts of target nucleic acid.
It would also represent a significant advantage if the method allowed the detection and quantification of several, preferably a large number such as 10 or more, polymorphic sites in a single reaction e.g. allowing the detection and quantification of several different target organisms in a single multiplex assay.
We have developed a convenient detection method which provides good sensitivity and specificity and the ability to detect and quantitate a large number of polymorphic sites in a single reaction. In particular, the methods represent significant advantages over those employing agarose gel electrophoresis or direct detection of the amplified DNA in the quantification.
The invention thus provides a method of detecting a target nucleotide sequence in a nucleic acid molecule, which comprises:
(a) binding of an oligonucleotide probe to said nucleic acid molecule;
(b) selective labelling of the bound oligonucleotide probe in the presence of said target nucleotide sequence;
(c) hybridisation of the labelled oligonucleotide to a complementary sequence; and
(d) subsequent detection of the label.
The method of the invention can be used in the detection of all target nucleotide sequences, such as DNA sequences, particularly DNA resulting from PCR amplification cycles. The DNA may be native or cDNA formed from mRNA by reverse transcriptase. The DNA may be single or double stranded, linear or circular. The target nucleic acid may be RNA, e.g. mRNA or in particular ribosomal RNA which is present in a cell in multiple copies, for example 3,000-20,000 copies per cell.
In the context of the present invention, the term xe2x80x9cnucleotide sequencexe2x80x9d may refer to a xe2x80x98sequencexe2x80x99 of only one nucleotide in length, where the nucleic acid of interest differs by only one nucleotide from other (non-target) sequences which it is not desired to detect, such as in the case of detection of a point mutation or polymorphism. More usually, the nucleotide sequence to be detected is characteristic of a particular nucleic acid or group of nucleic acids or of a particular organism or group of organisms e.g. a species, where it is desired to detect the presence of that target (e.g. the nucleic acid/or organism) in a sample containing a number of different molecules or organisms, such as in the detection of particular bacteria e.g. in a clinical or environmental sample.
The oligonucleotide probe may comprise 5 to 50, preferably 10 to 40, more preferably 20 to 30 nucleotides. The probe is selected to bind to the target nucleic acid, namely the nucleic acid molecule containing the target nucleotide sequence. The oligonucleotide is conveniently sufficiently large to provide appropriate hybridisation to the target nucleic acid, yet still reasonably short in order to avoid unnecessary chemical synthesis. Methods for oligonucleotide production are standard in the art.
As discussed above, the target nucleic acid may be DNA, cDNA or RNA. The oligonucleotide probe is designed to bind to a target region in the nucleic acid molecule containing the target nucleotide sequence. xe2x80x9cTarget regionxe2x80x9d is used herein to refer to the sequence of nucleic acid which binds to the oligonucleotide probe. This target region may be in the form of a xe2x80x9csignature sequencexe2x80x9d which characterises a particular nucleic acid or group of nucleic acids or organisms etc. or may be the section of nucleic acid immediately preceding a single base position of interest, if a polymorphism, point mutation, insertion or deletion is to be detected.
It is of course desirable that the target region be absent from any non-target molecules in the sample in order to introduce selectivity between target and non-target nucleotide sequences. This specificity of binding may be achieved by incorporating into the probe regions of complementarity or substantial complementarity to the desired target region and/or mismatches to non-target sequences. The region of complementarity may be present at terminal or internal segments of the probe, or both.
Conventionally, nucleic acid detection methods have involved labelling of the oligonucleotide probe prior to hybridisation to the target nucleic acid sequence. However, in our method, labelling of the probe is dependent upon the sequence of the nucleic acid molecule to which the probe is bound. In other words labelling of the probe is template-sequence dependent (i.e. sequence-specific)xe2x80x94incorporation of the label will is only occur in the presence of a desired or selected xe2x80x9csequencexe2x80x9d in the target molecule, which may be in the target region or in the target nucleotide sequence itself, or it will occur selectively ie. the step of label incorporation into the oligonucleotide probe may be discriminating (ie. based on a xe2x80x9cmini-sequencingxe2x80x9d principle). This adds an extra dimension of selectivity (specificity) to the process; oligonucleotide probe binding will initially discriminate against non-target nucleic acid but some binding to non-target sequences may be expected. If the probe itself were labelled prior to binding, then further discrimination would not be possible.
The present invention, on the other hand, adds a step of selectively (sequence-specifically) incorporating a label, in the presence of the target nucleotide sequence, into the oligonucleotide probe. This selectivity may be achieved, for example, by incorporation of the label only when the target nucleotide sequence is present, or by incorporating the label in a selective or discriminating manner such that the target nucleotide sequence may be discriminated, or both.
Conveniently, selective labelling may be achieved using labelled nucleotides ie. by incorporating (or not incorporating) into the oligonucleotide probe a nucleotide carrying a label. In other words selective labelling may occur by chain extension of the oligonucleotide probe using a polymerase enzyme which incorporates a labelled nucleotide. In the case of an RNA target, the polymerase will be a reverse transcriptase enzyme. Conditions for chain extension (ie. base incorporation) and suitable polymerase enzymes are well known in the art and widely described in the literature.
Selective labelling of the oligonucleotide is preferably achieved by incorporation of a labelled dideoxynucleotide.
The term dideoxynucleotide as used herein includes all 2xe2x80x2-deoxynucleotides in which the 3xe2x80x2-hydroxyl group is absent or modified and thus, while able to be added to the oligonucleotide probe in the presence of a polymerase, is unable to enter into a subsequent polymerisation reaction. This means that only one base can be added to the probe.
Chain termination in this manner allows the reaction to be controlled, and the assay to be quantitated, if desired (see later).
Advantageously, as described above, the present invention involves chain extension of the oligonucleotide probe to incorporate a label. The present invention permits selective labelling to be achieved both by incorporation of a labelled nucleotide in a selective manner, (ie. discriminating on the basis of the label, either its presence or absence, or by selective detection of a particular label) or by nucleotide incorporation in a selective manner such that incorporation of a nucleotide (whether labelled or not) occurs only in the presence of the target nucleotide sequence (or more accurately, that the efficiency of incorporation is much reduced where the target nucleotide sequence is not present) (ie. discriminating on the basis of nucleotide incorporation or non-incorporation), or both.
As an example of such a method, if the target sequence is a key differentiating base, e.g. if the base in the nucleic acid molecule immediately adjacent to the target region to which the probe binds, is a cytosine residue, then labelled dideoxyguanine triphosphate (ddGTP) would be added to the reaction mixture for incorporation. If the probe had bound to nucleic acid at a non-target sequence, then it is unlikely that the next nucleotide is the same as the key base, and the labelled ddGTP is then unlikely to be incorporated in the oligonucleotide ie. incorporation of the oligonucleotide takes place only (or largely or substantially only) in the presence of target. The above method of differentiation applies mutatis mutandis if the key base is guanine, adenine, thymine or uracil.
In order further to reduce the likelihood of misincorporation of the labelled dideoxynucleotide, unlabelled dideoxynucleotide triphosphates may also be added to the reaction mixture. Using the above example, where labelled ddGTP is added, unlabelled ddCTP, ddATP and ddTTP would be added (where the target nucleic acid molecule is DNA). If only one ddNTP is present, then despite the rules of base pairing, there may be a risk that it will be incorporated where the key base is not its natural base pair, albeit at a low frequency. This may cause a background or noise signal in the assay. The inclusion of the other ddNTPs means that these will be incorporated preferentially, according to the normal rules of base pairing (C-G and A-T/U), and misincorporation of the labelled ddNTP is significantly reduced. The use of unlabelled ddNTPs thus comprises a preferred embodiment of the present invention.
In a further preferred embodiment of the present invention, selectivity of the labelling reaction is further increased by incorporation of mismatches immediately upstream of the key base. Thus, the oligonucleotide probe is designed with a mismatch or mismatches at the 3xe2x80x2-end to non-target nucleotide sequences, and the presence of the mismatch(es) will reduce the likelihood of oligonucleotide extension. Therefore, even if the next base after the end of the bound oligonucleotide is by chance the same as the key base, in non-target sequences incorporation of the labelled ddNTP will be reduced where there is preceding mismatch between the probe and the nucleic acid to which it has bound.
A large number of suitable labels and labelling methods are known in the art and described in the literature. Any detectable or signal-generating label or reporter molecule may be used. Convenient labels include calorimetric, chemiluminescent, chromogenic, radioactive and fluorescent labels, but enzymic or antibody-based labelling methods or signal-generating systems may also be used. Thus the term xe2x80x9clabelxe2x80x9d as used herein includes not only directly detectable signal-giving moieties, but also any moiety which generates a signal or takes part in a signal generating reaction. Fluorescein or other fluorescently labelled ddNTPs are particularly suitable, which allow detection directly by fluorescence or indirectly by antibody interactions. These are commercially available from e.g. NEN/Dupont. ddNTPs can be labelled by e.g. [35S], [3H] or 32p] as described in Syvxc3xa4nen, A. C. et al. Genomics 8, [1990], 684-692.
In order to enhance sensitivity, more than one labelling step may be performed. In other words, the selective labelling step may be repeated one or more times, ie. performed cyclically. Increasing the number of cycles increases the sensitivity, and also when the assay is performed quantitatively (see below), the quantitative range of the method, by improving the label saturation of the probes. In order to facilitate cyclic labelling, a thermostable polymerase enzyme may be used in the label-incorporation step. Conveniently, the number of cycles of the selective labelling step may be from 1 to 50, e.g. 10 to 30 but this may be varied according to choice. An appropriate or convenient number of cycles for a given system may be determined by routine experiments.
Minisequencing methods to detect multiple single-nucleotide polymorphisms are known which involve single-nucleotide extension reactions with fluorescently labelled dideoxynucleotides (Pastinen, T. et al. Clinical Chemistry (1996) Vol. 42, 9, 1391-1397). In this case, the minisequencing reaction products were analysed by gel electrophoresis. In contrast, the present method involves a hybridisation reaction between the labelled oligonucleotide and its complementary nucleic acid sequence prior to detection and analysis.
Thus, following the selective labelling step(s), the labelled oligonucleotide probe is hybridised to a complementary nucleotide sequence. Generally, this involves separation of the labelled oligonucleotide probe from the previous reaction mixture. Label incorporation by incorporation of a labelled nucleotide, results in the so-labelled oligonucleotide probe hybridised to the target nucleic acid molecule. Strand separation to separate the probe may be achieved using denaturation methods conventional in the art e.g. high temperature heating, treatment with high pH (alkali) etc. Step (c), hybridisation to a sequence complementary to the probe, may then take place. This xe2x80x9ccomplementary sequencexe2x80x9d according to the invention may be any nucleotide sequence containing or comprising a region of absolute or substantial complementarity to the probe such that binding of the probe to the xe2x80x9ccomplementary sequencexe2x80x9d can occur. The complementary sequence may be DNA or a modification thereof such as PNA. Conveniently, the complementary sequence may take the form of the complement of the probe (ie. a sequence fully complementary to the probe). It will be understood however, that this is not absolutely essential and that the xe2x80x9ccomplementxe2x80x9d may be contained in a longer sequence, may be partial or truncated, and/or may contain sequence variation (ie. less than perfect complementarity) as long as binding of the probe to the xe2x80x9ccomplementary sequencexe2x80x9d may still occur.
In a preferred embodiment, the complementary sequence is immobilised on a solid support. Suitable immobilising supports to which the complements can be attached are known in the art and include any of the well known supports or matrices which are currently widely used or proposed for immobilisation, separation etc. These may take the form of particles, sheets, gels, filters, membranes, fibres, capillaries, chips or microtitre strips, tubes, plates or wells etc. Methods of immobilising or attaching oligonucleotides to solid supports are likewise known in the art. Particularly preferred are membrane strips (commercially available from NEN and known as GeneScreen) on to which the complementary sequences may be spotted and then U. V. cross-liked, or DNA chips (microchips, glass chips) now common in molecular biology procedures. The use of such solid supports, in particular chips which can carry an oligonucleotide array ie. a number, e.g. up to 250, of different oligonucleotides complementary to the oligonucleotide probes used in the earlier part of the assay, represents a particularly preferred embodiment of the invention.
Hybridisation step (c) permits the quantitative separation of the labelled probes. This is advantageous in the context of a quantitative assay.
Following the hybridisation step, the label on the oligonucleotide probe is detected. As mentioned above in the context of the label, the detection may be by any means known in the art, depending on the label selected. Signal amplification may also be used, if desired, as described in the art (see, for example, P. Komminoth and M. Werner, Target and Signal Amplification, approaches to increase the sensitivity of in situ hybridisation, 1997 Histochem. Cell Biol. 108:325-333). Detection may also be qualitative, or quantitative or semi-quantitative. Thus, for example, a visual e.g. calorimetric, chromogenic, fluorescent or other signal detectable from the label may be used to provide a simple yes or no indication of the presence or absence of the target nucleotide sequence. Advantageously, however, the detection is such as to yield a quantitative assessment of the amount of target nucleotide present in the sample. This may be an absolute indication of the amount of target present or some other quantitative or semi-quantitative indicator e.g. a percentage, ratio, or similar indicator, e.g. relative to total nucleic acid in the sample.
Quantitation of the target nucleotide sequence may further be correlated to a quantitation of the number of organisms or cells containing the target in question. Thus, in further aspects, the invention provides methods of determining the amount of a target nucleotide sequence or the number of cells containing a target nucleotide sequence using the nucleic acid detection assay hereinbefore described.
As mentioned above, the target nucleic acid may be any DNA or RNA molecule. It may thus be directly the nucleic acid of a target cell or organism. It may be desirable, however, to amplify the nucleic acid prior to detection using the assay method of the invention and advantageously the target nucleic acid may thus be an amplicon of an in vitro amplification reaction. Amplification may however also take place by other means including in vivo cloning methods. Any method of in vitro amplification may be used, including both linear and exponential methods. Representative methods thus include PCR, NASBA and ligase chain reaction. Due to its convenience, PCR and its variants will commonly be the method of choice.
In the context of a quantitative assay, a quantitative in vitro amplification method may be combined with the detection assay of the invention. In particular, in a preferred embodiment of the methods of the present invention, the oligonucleotide probe binding step is preceded by a competitive PCR reaction. A variety of methods of competitive PCR exist and are described in the literature. Any of the known techniques may be used. (For a review see e.g. Srebert, P. D. and Larrick, J. W. [1992] Nature, 3, 557-558).
In such a method, nucleic acid incorporating the target nucleotide sequence is co-amplified with a selected competitor molecule containing the same primer template sequences as the target molecule and thus the competitor competes with the target for PCR primer binding and amplification. The competitor comprises a means for differentiating from the target, conveniently a recognition sequence for binding to a competitor-specific oligonucleotide probe, analogous to the xe2x80x9ctarget-specificxe2x80x9d oligonucleotide probe described in the context of the detection assay above (ie. the probe of step (a) above). The recognition sequence is thus analogous to the xe2x80x9ctarget regionxe2x80x9d of the oligonucleotide probe above. A single known amount of competitor may be added, for comparison to a standard curve, or a series of amplifications may be performed using varying amounts of competitor e.g. a dilution series. Primers complementary to sequences flanking the target nucleotide sequence in the nucleic acid molecule of interest (and to the same sequences in the competitor) are added and PCR amplification is performed. Competitive PCR enables the detection to be performed with a built in reference to provide more accurate analysis of the results. A standard curve may be constructed using standard techniques conventional in the art.
In such a competitive PCR step, amplicons are produced deriving from both target and competitor DNA. The competitor amplicon is subjected to a detection assay procedure in an analogous manner to the target amplicon, as described above. The amplified competitor sequence includes a known recognition sequence which behaves in the same way as the target region in the target nucleic acid molecule under detection. For example, an oligonucleotide probe binding selectively to this recognition region of the competitor is added to the reaction mixture at the same time as the probe to the target region of the target molecule is added, both oligonucleotides being designed to be single base extended with a labelled ddNTP when bound to their respective target sequences. A complementary sequence to the competitor oligonucleotide probe which has been labelled following binding to the competitor is also provided, preferably immobilised, and the labelled oligonucleotide probe hybridises to this complementary sequence before detection. The labels of the target and competitor oligonucleotides can then be detected and, in particular, the relative strengths of the measured signal used to evaluate the amount of target nucleic acid which was present in the original sample given the known amount of competitor added to the system. This aspect of the invention is represented in FIG. 1. It is not necessary that the label for the competitor and the target sequence are different, indeed they are preferably the same. Differentiation between competitor and target, as between a plurality of different target sequences, is preferably achieved spatially; the position of the complementary sequence to each oligonucleotide probe on e.g. the microchip array is known and the strength of signal at each position is measured.
Titration experiments can be used to determine the optimum amount of competitor to be used in any particular assay, as described in the following Examples. The amount of competitor selected preferably allows detection of a low frequency of target nucleic acid molecules and reproducible amplifications. Suitable concentrations may vary from 6xc3x9710xe2x88x928 to 6xc3x9710xe2x88x9210 pmol, preferably from 4xc3x9710xe2x88x929 to 8xc3x9710xe2x88x929 pmol.
Preferably, sufficient cycles of PCR are performed so that a saturation level is achieved; in that way the amplified DNA contains substantially the same ratio of target DNA to competitor DNA as that present before PCR.
It is advantageous to provide conditions such that the competitor and target DNA are amplified equally efficiently by PCR and it is therefore desirable that the size and GC-content of each fragment is kept substantially the same. In some instances it will also be necessary to take into account the efficiency of the cell lysis of the sample which can make some of the genomic DNA less available for the PCR. In addition, the target DNA may be part of chromosomal DNA in the initial cycles, while the competitor DNA may be, for example, in the form of a small plasmid. Thus it might be necessary to adjust for the relative amplification efficiencies of these early PCR cycles.
Alternative amplification strategies may also be used in this aspect of the invention, for example NASBA and ligase chain reaction.
Any suitable polymerase may be used, although it is preferred to use a thermophilic enzyme such as Taq polymerase to permit the repeated temperature cycling without having to add further polymerase, e.g. Klenow fragment, in each cycle.
As mentioned above, the target nucleic acid may be cDNA synthesised from mRNA in the sample, and the method of the invention is thus applicable to diagnosis on the basis of characteristic mRNA. Such preliminary synthesis can be carried out by a preliminary treatment with a reverse transcriptase, conveniently in the same system of buffers and bases to be used in the subsequent amplification (e.g. PCR) steps. Since the PCR procedure requires heating to effect strand separation, the reverse transcriptase will be inactivated in the first PCR cycle. When mRNA is the target nucleic acid, it may be advantageous to submit the initial sample, to treatment with an immobilized polydT oligonucleotide in order to retrieve all mRNA via the terminal polyA sequences thereof. Alternatively, a specific oligonucleotide sequence may be used to retrieve the RNA via a specific RNA sequence. The oligonucleotide can then serve as a primer for cDNA synthesis, as described in International Patent Application WO-A-90/11446.
The PCR (or other in vitro) amplification of target and competitor nucleic acid provides a first level of specificity but a low level of non-target/non-competitor molecules may be amplified in this reaction, hence the selective labelling of the oligonucleotide probes based on signature sequences in the target and the competitor amplicons according to the present invention. The quantification is based on the signal ratio of target to competitor alone and is not affected by any non-target/non-competitor molecules which may have been amplified in the first reaction.
When the main detection assay follows a PCR or other in vitro amplification step based on chain extension, then it is necessary to separate the dNTPs from the previous reaction e.g. in the PCR product or to inactivate them to ensure that they could not be involved in subsequent elongation of the oligonucleotides bound to target/competitor nucleic acid in place of the terminating ddNTPs. Physical separation of the dNTPs from the PCR product is possible by e.g. gel electrophoresis, precipitation, affinity purification, chromatography or filtration. However it is preferred to inactivate the dNTPs by removing the reactive phosphate groups by a phosphatase so that the nucleotides can no longer be used for significant chain elongation.
Target nucleic acid for use in the method of the invention may be obtained or prepared by any convenient technique in the art. Many different methods for isolating nucleic acids from cells and organisms are described in the literature and any of these could be used.
For example, methods for the extraction of nucleic acid from environmental samples are described by Bowman, J. P. and Sayler, G. S. in Molecular Approaches to Environmental Microbiology (1996), pp 63-97. However, a particularly preferred embodiment of the present invention combines a detection method as described above with the solid-phase method for combined cell and nucleic acid isolation described by us in Rudi et al., Appl. Environ. Microbiol. (1998), 64(1), pp 34-77.
The method of isolating nucleic acid from a sample of cells which is described in this paper comprises:
(a) binding cells in the sample to a solid support to isolate the cells from the sample;
(b) lysing the isolated cells; and
(c) binding nucleic acid released from said lysed cells to said same solid support.
The bound nucleic acid may be used directly in a detection assay of the invention or it may be released from the support.
The detection assay of the present invention may advantageously find application in any procedure where it is desired to detect nucleic acids, for example in any procedure relying on DNA or RNA identification, e.g. diagnosis, detection of infections or contaminating organisms or pathogens, clinical monitoring, forensics, food and environmental safety and monitoring, or for distinguishing between different cells or cell types, mutation detection, analysis of polymorphisms, e.g. in tissue typing etc. and as a general molecular biology tool. The complete nucleic acid-based assay comprising sample preparation, DNA amplification, selective labelling and detection is of particular benefit in quantifying the amount of toxin-producing cyanobacteria in water sources. This combined isolation and detection method comprises a particularly preferred embodiment of the present invention. The assay provides a lower detection limit of 200, preferably 100 cells/ml (of a particular cyanobacteria against a background of 105 cells/ml of other cyanobacteria) and a quantitative range of more than three orders of magnitude. The methods described in principle above and in more detail below are suited for automation, providing the means for the development of high throughput systems for routine environmental monitoring.
For example, water-blooms formed by cyanobacteria have a relatively high frequency of toxicity (between 25 and 70%), and constitute a potential health hazard to livestock and humans. This is particularly the case for blooms of cyanobacteria belonging to the genus Microcystis. Species of this genus can produce several toxins, with the hepatotoxic microsystins being the most potent. It would therefore be desirable if a method could be developed which provided a reliable, quick and convenient way to detect and quantify these bacteria.
The methods of the invention are particularly suitable in this respect, in that by selection of oligonucleotides to signature sequences within the genome of different bacteria of interest, a single sample can be analysed in one procedure for several different microorganisms. Signature sequences for distinct bacterial groups are widely known and published in the literature, for cyanobacteria a classification system based on 16S rDNA sequence information can be used (see e.g. J. R. Marchesi, T. Sato, A. J. Weightman, T. A. Martin, J. C. Fry, S. J. Hiom, and W. G. Wade, 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64:795-799: Turner, S. (1998) Origins of algae and their plastids, pp 13-53 Ed. Bhattacharya, D. NY: Springer-Verlag and Giovanni, S. J., Turner, S., Olsen, G. J., Barns, S., Lane, D. J. and Pace, N. R. (1988) J. Bacteriol 170 pp 3584-92).
The Examples which follow describe a fully integrated assay for analysis of complex cyanobacterial communities. The methods of the invention are equally suitable for use in the investigation of other microbial communities.
Complementary sequences to the different labelled oligonucleotides can be arranged on a single microchip in a pre-determined manner to allow quantification of the levels of different bacteria, preferably by comparison of the ratio of competitor signal to the signal for each bacterial group. Such multiplex quantitative assays preferably use high density oligonucleotide arrays immobilised on glass chips, with subsequent direct detection of the fluorescein-label (see e.g. Schena, M. et al., Science, (1996) 270: 467-470; and R. J. Lipshutz, D. M. Morris, M. Chee, E. Hubbell, M. J. Kozal, N. Shah, N. Shen, R. Yang, and S. P. A. Fodor. Using oligonucleotide probe arrays to access genetic diversity, Bio Techniques 19:442-447).
The invention also comprises kits for carrying out the methods of the invention. These will normally include at least the following components:
(a) an oligonucleotide probe capable of binding to a target nucleic acid molecule containing the target nucleotide sequence (ie. directed to a target region within the nucleic acid molecule;
(b) means for selective labelling of the oligonucleotide probe; and
(c) nucleotide sequences complementary to the oligonucleotide probes, preferably immobilised on a solid support.
The means for selective labelling will conveniently comprise a polymerase enzyme and at least a labelled nucleotide, preferably a labelled ddhTP, optionally with corresponding unlabelled nucleotides also present.